The proposed research explores the structure and mechanism of terpenoid cyclases, which are unique among enzymes in that they catalyze the most complex carbon-carbon bond forming reactions in biology: on average, more than half of the substrate carbon atoms undergo changes in bonding and/or hybridization during the course of a typical enzyme-catalyzed reaction. Notably, many terpenoids exhibit useful pharmacological properties, such as the blockbuster cancer chemotherapy drug Taxol (paclitaxel) and the antimalarial drug artemisinin. Thus, a better understanding of terpenoid cyclase structure and mechanism will enable drug discovery and manufacture at the interface of natural products chemistry, enzymology, structural biology, and synthetic biology. To advance our understanding of structure-function relationships in terpenoid cyclases, we will pursue the following lines of investigation: (1) We will determine the structural basis of reprogrammed cyclization cascades catalyzed by site-specific mutants of the C15 sesquiterpene cyclase epi-isozizaene synthase from Streptomyces coelicolor (EIZS). We will study the temperature dependence of cyclization fidelity in selected EIZS mutants and we will determine crystal structures of complexes with analogues of substrate and carbocation intermediates, as well as the C15 hydrocarbon product. These snapshots of wild-type and mutant EIZS cyclization cascades will show us how cyclization chemistry can be redirected to generate alternative products. (2) We will determine the structural basis of cyclization fidelity in the bifunctional C20 diterpene cyclase fusicoccadiene synthase from Phomopsis amygdali (PaFS). We will study the temperature dependence of PaFS cyclization fidelity and we will determine crystal structures of complexes with substrate analogues and the hydrocarbon product to generate snapshots of the diterpene cyclization cascade. We will also study a double mutant designed to introduce C25 sesterterpene cyclase activity by increasing active site volume. These structures will be the first to map out the reaction coordinate of a diterpene cyclization reaction. (3) We will determine the structural basis of assembly-line biosynthesis in the bifunctional C25 sesterterpene synthases ophiobolin F synthase from Aspergillus clavatus (AcOS) and mangicdiene synthase from Fusarium graminearum (FgMS). We will develop a radiolabeled substrate to measure the steady-state kinetics of sesterterpene hydrocarbon formation, and we will determine whether channeling occurs between the prenyltransferase and cyclase active sites of each bifunctional enzyme. We will also determine structures of AcOS and FgMS using X-ray crystallography and/or electron microscopy to better understand assembly-line terpenoid biosynthesis. These studies will provide the first structural views of sesterterpene synthases, a family of terpenoid biosynthetic enzymes discovered only recently.